Download Identification of a new monosaccharide in Mycobacterium

Identification of a new monosaccharide in Mycobacterium tuberculosis
lipoarabinomannan
Bruce Turnbull, Kazumi Shimizu, Achim Treumann, Susanne Hesketh and Steve Homans
Introduction
Tuberculosis (TB) remains a major threat to world health, with approximately eight million
cases of TB resulting in two million deaths per annum. Ninety-five percent of TB cases arise
in the developing world, where the HIV epidemic has dramatically increased the number of
people who succumb to the disease. The WHO has highlighted the urgent need for more
effective anti-TB drugs and for rapid, specific and sensitive diagnostic tests for the causative
agent, Mycobacterium tuberculosis (Mtb). The ability of the Mtb to enter and colonise
macrophage cells can, in part, be attributed to a carbohydrate structure called
lipoarabinomannan (LAM, Fig. 1a), that is present in the Mtb cell wall. Mannose residues
that cap the dendritic arabinan chains of LAM, facilitate entry of Mtb into alveolar
macrophages, following interaction with the macrophage mannose receptor. LAM then
promotes the intracellular survival of Mtb by down-regulating the immune response and
providing anti-oxidative protection for the bacterium. The mechanisms by which LAM
exerts is biological activity inside macrophage cells remain to be elucidated.
Fig. 1. (a) Cartoon representation of LAM showing the location of MTX/MSX (red) attached to the mannosyl
caps (green) at the ends of the arabinan (blue) chains. (b) An overlay of HSQC spectra for the α-methyl
glycosides of MTX (red), and MSX (grey) and LAM (black). (c) Inset is a strip from an HCCH-TOCSY
spectrum of LAM which highlights the 1H chemical shifts for MTX and MSX that correlate with the anomeric
signals at ca. 5.4 ppm
Recently, we discovered an unusual sugar residue attached to the mannosyl caps of LAM.
High resolution NMR spectroscopy and mass spectrometry studies revealed that this novel
monosaccharide was a 5-methylthio-pentofuranoside, which was partially oxidised to the
sulfoxide. However, it was not possible to determine the stereochemistry of the pentose
using NMR methods. Consequently, we chemically synthesised all eight possible isomers of
methylthio-pentose in the form of their methyl glycosides. On comparing the HSQC spectra
for these compounds with that for LAM, it became clear that only the α-xylo-configured
sugar (methylthio-xylofuranoside, MTX) gave signals that correlated with the natural product
(Fig. 1b and c). Similarly, the chemical shifts for the sulfoxide form of the natural sugar were
in generally good agreement with those for the oxidised α-xylofuranoside (MSX).
The discovery of MTX constitutes the first example of a methylthio-sugar residue
incorporated into a polysaccharide, and only the fifth example of a xylo-configured sugar
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outside the plant kingdom. Presumably, the biosynthetic precursor of MTX is methylthioadenosine – a ubiquitous by-product of polyamine biosynthesis that is derived from SAM.
Nevertheless, Mtb invests significant biosynthetic effort into incorporating MTX into its cell
wall, which implies that this sugar may provide some advantage to the bacterium. The
occurrence of MTX at the non-reducing termini of the polysaccharide, locates it at the
bacterium’s surface, where it could easily act as a ligand for receptors or regulatory proteins,
either on the surface of the macrophage, or inside the host cell, following endocytosis. It is
possible that conversion of MTX to its oxidised form (MSX) may even play a role in LAMmediated oxidative protection for Mtb. Further structural and biochemical studies of MTX
are in progress.
Collaborators
Delphi Chatterjee, Department of Microbiology, Immunology and Pathology, Colorado State
University, USA.
Publications
Turnbull, W.B., Shimizu, K.H., Chatterjee, D., Homans, S.W. and Treumann, A. (2004)
Identification of the 5-methylthiopentosyl substituent in Mycobacterium tuberculosis
lipoarabinomannan. Angew. Chem. Int. Ed. 43, 3918-3922.
Funding
This work has been funded by the Wellcome Trust and the University of Leeds.
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